Pfx accuprime master mix

Fecal samples were thawed on ice and homogenized. Then 1ml of each sample was used for DNA extraction using the QIAamp PowerFecal DNA kit according to the manufacturer’s instructions (Qiagen, United States). To understand the bacterial community, a dual-index sequencing strategy was applied (Kozich et al., 2013 (link)). Briefly, the V4 region of the 16S rRNA gene was amplified using the PCR with dual-index primers (Kozich et al., 2013 (link)). The PCR amplification reaction included 1μl forward index primer (10mM), 1μl reverse index primer (10mM), 1μl 10ng/μl DNA template, and 17μl Pfx AccuPrime master mix (Invitrogen, United States). Amplification was initiated with denaturation for 5min at 95°C, followed by 30cycles of 95°C for 30s, annealing at 55°C for 30s and extension at 72°C for 1min, with a final elongation for 5min at 72°C. The amplicons were then purified and normalized using the SequalPrep plate normalization kit (Invitrogen, United States). The same amount of barcoded V4 amplicons from each sample was pooled to construct the DNA library.

The V4 hypervariable region of bacterial 16S rRNA gene was amplified using dual-index primers (Caporaso et al., 2011 (link)) according to Kozich et al. (2013) (link). The PCR amplification reaction consisted of 1 µL forward index primer (10 mM), 1 µL reverse index primer (10 mM), 1 µL DNA template (10 ng/µL), and 17 µL Pfx AccuPrime master mix (Invitrogen, USA). The reaction protocol consisted of denaturation for 5 min at 95 °C, followed by 30 cycles of 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 1 min, and elongation for 5 min at 72 °C. The amplicons were run on a 1% agarose gel to confirm success of the PCR and normalized with a SequalPrep Normalization Plate Kit (Applied Biosystems, Foster City, CA) to construct the DNA pool library. A total of 64 samples were sequenced at the Interdisciplinary Center for Biotechnology Research (ICBR) of the University of Florida using a MiSeq reagent kit V2 (2 × 250 cycles run; Illumina, San Diego, CA, USA) in an Illumina MiSeq platform (Illumina, San Diego, CA, USA). Sequencing data were deposited into the NCBI database with the following accession number PRJNA854650.

The V4 variable region of bacterial 16S rRNA gene was amplified using dual-index primers (Caporaso et al., 2011 (link)) according to Kozich et al. (2013) (link). The PCR amplification reaction consisted of 1 µL forward index primer (10 mM), 1 µL reverse index primer (10 mM), 1 µL DNA template (10 ng/µL), and 17 µL Pfx AccuPrime master mix (Invitrogen, USA). The protocol for the reaction was the following: denaturation for 5 min at 95 °C, followed by 30 cycles of 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 1 min, and elongation for 5 min at 72 °C. Resulting amplicons were run on a 1% agarose gel to confirm success of the PCR and normalized with a SequalPrep Normalization Plate Kit (Applied Biosystems, Foster City, CA) for construction of the DNA pool library. Overall, 32 samples (16 samples of the liquid effluent fraction and 16 samples of the solid effluent fraction) were sequenced at the Interdisciplinary Center for Biotechnology Research (ICBR) of the University of Florida using a MiSeq reagent kit V2 (2 × 250 cycles run; Illumina, San Diego, CA, USA) in an Illumina MiSeq platform (Illumina, San Diego, CA, USA). The 16S rRNA gene amplicon sequencing data were deposited into the NCBI database (accession number PRJNA811164).